Single-Molecule Imaging of Cytoplasmic Dynein Reveals the Mechanism of Motor Activation and Cargo Capture

bioRxiv preprint doi: https://doi.org/10.1101/2021.04.05.438428; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Single-molecule imaging of cytoplasmic dynein reveals the mechanism of motor activation and cargo capture

Nireekshit Addanki Tirumala1 and Vaishnavi Ananthanarayanan1,2,

1Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore, India 2Current afﬁliation: EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Australia

Cytoplasmic dynein 1 (dynein) is the primary minus end- 1a, see Methods). We observed that dynein spots appeared directed motor protein in most eukaryotic cells (1). Dynein re- afresh and remained in the field of imaging for a short dura- mains in an inactive conformation until the formation of a tri- tion (Fig. 1b, Supplementary Video 1). We intuited that these partite complex comprising dynein, its regulator dynactin and a intensity traces corresponded to events where single dynein cargo adaptor (2–5). Thereupon, dynein transports cargo to- molecules previously diffusing in the cytoplasm bound to wards the minus ends of microtubules. How this process of MTs (Extended Data Fig. 1c-e). motor activation occurs is unclear, since it entails the formation of a three-protein complex inside the crowded environs of To confirm that the appearance of fluorescent signal on the a cell. Here, we employed live-cell, single-molecule imaging MT corresponded to binding of a single molecule of dynein to visualise and track fluorescently tagged dynein. First, we to the MT, we analysed the intensity of these fluorescent spots observed that dynein that bound to the microtubule engaged (Fig.1c). For single dynein molecules, we would expect the in minus end-directed movement only ~30% of the time and intensity histogram to fit to a sum of two Gaussian distribu- resided on the microtubule for a short duration. Next, using tions, one corresponding to a GFP fluorescing from one DHC high-resolution imaging in live and fixed cells, we discovered and the other corresponding to two GFPs fluorescing from that dynactin remained persistently attached to microtubules, both DHCs in the genetic background of these cells. The for- and endosomal cargo remained in proximity to the microtubules mer primarily arose due to photobleaching of GFP during the and dynactin. Finally, we employed two-colour imaging to visu- course of imaging. Accordingly, the intensity histogram of alise cargo movement effected by single motor binding. Taken these fluorescent spots revealed that we indeed observed sin- together, we discovered a search strategy that is facilitated by dynein’s frequent microtubule binding-unbinding kinetics: (1) gle dynein molecules since the intensity histogram fit best to in a futile event when dynein does not encounter cargo anchored a sum of 2 Gaussians, with the mean of the first being half in proximity to the microtubule, dynein unbinds and diffuses that of the second (Fig. 1c). into the cytoplasm, (2) when dynein encounters cargo and dyn- Next, we analysed the x, y vs t tracks of single molecules actin upon microtubule-binding, it moves cargo in a short run. of dynein that bound afresh from the cytoplasm to the MT In conclusion, we demonstrate that dynein activation and cargo (Fig. 1d). Based on automated thresholding (see Methods), capture are coupled in a step that relies on reduction of dimen- we classified the tracks as stationary, minus end-directed sionality to enable minus end-directed transport in vivo. and plus end-directed. The plus ends of the MT were pre- cytoplasmic dynein | dynactin | cargo trafficking | single-molecule imaging dominantly at the periphery in these elongated cells (Ex- Correspondence: [email protected] tended Data Fig. 1f), and hence we annotated movement towards the cell center as minus end-directed, and movement away, plus end-directed. We observed that ~50% of Results and Discussion all the dynein molecules tracked (n=177/329, N=3 indepen- Dynein interacts transiently with the microtubule dent experiments from >50 cells) remained stationary upon To visualise the behaviour of dynein in HeLa cells express- MT binding, while ~30% (n=95/329) moved towards the mi- ing mouse DYNH1C1-GFP (mDHC-GFP, (6)), we adapted nus end (Fig. 1e). The remaining ~20% moved towards the and optimised highly inclined and laminated optical sheet plus end and arose likely due to attachment of dynein to cargo (HILO) microscopy ((7), Extended Data Fig. 1a). When cells being moved to the plus end by kinesins (Fig. 1e). The ve- expressing low levels of mDHC-GFP were observed under locity measured for minus-end directed movement of single a spinning disk confocal (SD) microscope, the fluorescence dyneins was 1.2 ± 0.7 µm/s (mean ± s.d.), similar to values signal appeared cytosolic, with no discernible dynein punc- reported for mammalian dynein previously (10, 11). We also tae (Extended Data Fig. 1b). However, when the same cells confirmed that the underlying MT was stable, did not un- were observed using our modified HILO microscopy, distinct dergo sliding, and therefore did not contribute to the dynein fluorescent spots were visible. behaviour we observed (Extended Data Fig. 1g). We adapted our microscopy protocol to obscure dynein dif- We then measured the mean residence time of dynein on fusing in the cytoplasm and to only observe dynein that the MTs to be ~0.55s (95% confidence interval (CI): 0.51- bound to and resided on the microtubule ((8, 9), MT, Fig. 0.59s) (Fig. 1f). We verified that this short residence time of

Tirumala et al. | bioRχiv | April 5, 2021 | 1–8 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.05.438428; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig. 1. Visualisation of single molecules of dynein in living cells. a, Schematic of the protocol followed for the visualisation of single molecules of dynein. b, Montage of HILO images showing representative binding and unbinding events of a single fluorescent mDHC molecule (‘(binding)’ and ‘(unbinding)’). The single molecule is indicated with yellow arrowheads for the duration of the time it remains bound in the field of view. Time is indicated at the top right of each image in the montage. c, Intensity histogram of single molecules of dynein with the Gaussian fits (grey line). The mean ± s.d. of the Gaussian distributions is indicated above the fits. d, HILO image (left) and kymograph (right) of a cell expressing mDHC-GFP.Representative stationary, minus end-directed and plus end-directed events are indicated with the white, teal and magenta arrowheads respectively in the kymograph. e, Plot of displacement vs. time for the single-molecule events tracked, showing stationary events (grey), minus end-directed events (teal) and plus end-directed events (magenta). f, Histogram with the residence time of dynein on the MT on the x axis and 1-cumulative frequency on the y axis. The exponential fit (grey line) gave a mean residence time (‘τ’) of ~0.55 s. In b and d, ‘N’ marks the location/direction of the nucleus. dynein on MTs was a true representation of the duration of absence of the cargo adaptor (2–5). time dynein remained attached to the MT and not convolved The +TIP protein EB1 has been found to recruit another +TIP by GFP’s photobleaching time (Extended Data Fig. 1h and protein, CLIP-170, which in turn binds and clusters dynactin i). Further, by knocking down endogenous HeLa DYNC1H1 via its p150 subunit at growing MT plus ends (17). MT (hDHC), we verified that our observations were not an arte- plus ends decorated with dynactin also accumulated dynein fact of expression of mDHC-GFP in this background (Ex- at these sites, and evidence suggested that cargo transport tended Data Fig. 2 a-h). To the best of our knowledge, these was initiated when these MT plus ends contacted intracel- are the first observations of single molecules of dynein in lular cargo (18, 19). However, MT plus end-mediated initia- mammalian cells and indicate that dynein likely exists in an tion of dynein-driven transport appears to vary with cell type inactive state inside the cell, similar to reports from in vitro and context (17, 20, 21). Therefore, using SD microscopy in studies (2–5). combination with super-resolution radial fluctuations (SRRF, Dynactin remains persistently associated with MTs (22)) we first quantified the localisation of p150 (Fig. 2a). Next, we aimed to visualise the dynamics of the second Our high-resolution images revealed that only ~17% of the player in the tripartite complex, dynactin. Dynactin was first MT plus ends were enriched with p150, and p150 appeared identified as a complex that was required for dynein-driven bound along the entire length of the MT lattice (Fig. 2b). Fur- motility of vesicles in vitro (12). Several recent pieces of ther, by quantifying the intensities of mDHC-GFP expressed research have identified dynactin as an essential part of the in our cells, we concluded that the significant MT plus end lo- active dynein complex (13–15). Dynactin is a multi-subunit calisation of dynein reported in earlier studies (2, 23) might complex which binds to MTs independently of dynein via its have been an artefact of dynein overexpression (Extended N-terminal p150 subunit (16). However, dynactin’s interac- Data Fig. 3a, b). tion with dynein has been observed to be improbable in the However, the dynactin complex has been observed to show

2 | bioRχiv Tirumala et al. | Dynein regulation in vivo bioRxiv preprint doi: https://doi.org/10.1101/2021.04.05.438428; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

lattice. Additionally, fluorescently tagged p150 had residence times on the MT that far exceeded that of dynein, with most spots of p150 remaining bound to the MT for the entire duration of the time-lapse video (~30 s) (Extended Data Fig. 3c, Supplementary Video 2). Further, we observed that depletion of p150 through siRNA mediated silencing reduced the levels of both p150 and p62 along the MT lattice (Extended Data Fig. 3d-i) To test that the perturbation of dynactin localisation along the MT lattice resulted in reduced dynein activity, we depleted p150 using siRNA mediated silencing. As expected, we observed a reduction in the proportion of dynein molecules that moved towards the MT minus ends with a con- comitant increase in the proportion that moved towards the plus ends (Extended Data Fig. 4 a-d). Endosomes remain close to MTs and move in short bursts We then sought to understand how dynein interacted with the third component of the active complex - the cargo adaptors. The endosomal cargo adaptors, Hook proteins, have been observed to remain persistently bound to their respective cargo (24, 25). Therefore, we used endosomal cargo as a proxy for the cargo adaptor. To avoid artefacts from overexpressing fluorescently tagged Rab5 to visualise early endosomes (26), we employed cells that had taken up 10kDa dextran conju- gated with Alexa 647 via fluid-phase endocytosis (Extended Data Fig. 5a).

Fig. 2. The dynactin complex binds along the entire length of the MT. a, Im- Following a short pulse and chase, we observed Rab5- munofluorescence images of MT (top left), p150 (bottom left) and their merge (right) positive fluorescent compartments (Extended Data Fig. 5b, obtained using SD microscopy + SRRF. b, Enlarged view of the area marked with Supplementary Video 3). We visualised dextran vesicles in the white rectangle in a (top) and the line profile of p150 intensity along the length of a representative MT from the plus end (‘P’) to ~6 µm from the plus end of the MT 10s-timelapses and observed that ~85% of them were im- (‘0’). c, Immunofluorescence images of p62 (green) and p150 (magenta) obtained mobile (Fig. 3a, Supplementary Video 4), with only ~15% using SD microscopy + SRRF. The white arrowheads indicate representative p150 of these vesicles moving >1 µm during this time (Fig. 3b). spots that also contain p62. d, Histogram of the probability of cooccurrence of p62 with p150, indicating a high likelihood of presence of the entire complex at a p150 Strikingly, the dextran vesicles displayed uninterrupted mi- spot. e, Immunofluorescence images of MT (magenta) and p62 (green) obtained nus end-directed runs that lasted only 0.56 ± 0.25 s (mean ± using SD microscopy + SRRF. The white arrowheads indicate representative p62 s.d.) on an average (Fig. 3c) (n=719 dextran vesicles from spots that occur on the MT. f, Histogram of the probability of cooccurrence of p62 on the MT, which points to a high likelihood for the presence of the entire dynactin N=1 independent experiment with > 30 cells). These results complex on the MT. In a, b, c and e, ‘N’ marks the location/direction of the nucleus. are comparable to the run-and-pause behavior observed for endosomal cargo in previous studies (10, 11). no MT-binding in the absence of dynein in in vitro assays Next, we probed the localisation of the dextran vesicles with (3). To ascertain that the p150 spots that we observed in these respect to the MT. First, we imaged dextran vesicles and MTs cells represented the entire dynactin complex, we used SRRF in live cells and observed that the vesicles were in proximity to visualise p150 in concert with another dynactin subunit, to the MTs (Fig. 3d). We also observed that the dextran vesi- p62 (Fig. 2c). The p62 subunit of dynactin is located in the cles remained close to MTs even while they had no apparent pointed end complex of dynactin (16), and colocalisation of tether to the MTs via motor proteins, and therefore stationary p62 with p150 would indicate the presence of the complete (Supplementary Video 5) in minute-long live cell time-lapse dynactin complex. We observed that 49 ± 12% (mean ± s.d.) images. Then, to understand if the dextran vesicles could of the p150 spots colocalised with p62 (n=21,934/44,306 diffuse away from their original locations on the MT upon spots from N=2 independent experiments with 59 cells, Fig. motor unbinding, we depolymerised MTs using nocodazole 2d). So too, we used SRRF to additionally visualise the local- and visualised movement of the vesicles thereafter (Extended isation of p62 on MTs. The presence of p62 on MTs would Data Fig. 5c, Supplementary Video 6). Confirming previous indicate association with the MT of a subunit which does not findings (10, 11), we measured a lower diffusion coefficient normally do so unless it is part of the entire dynactin com- for the vesicles in the absence of MTs (Extended Data Fig. plex. Therefore, occurrence of p62 on the MT would im- 5d), likely implicating high intracellular crowding in con- ply localisation of the entire complex on the MT via p150. straining vesicle diffusion (11). Finally, we used SRRF to We observed that 74 ± 18% (mean ± s.d.) of the p62 spots visualise Rab5 in concert with p62 in living cells, and ob- (n=59,715/79,639 spots from 1 experiment with 25 cells) served that ~70% of the Rab5 spots colocalised with p62 were present on MTs (Fig. 2e, f). Therefore, the complete (n=8,795/12,848 vesicles from N=2 independent experiments dynactin complex is likely present along the length of the MT with >20 cells each), indicating that cargo and dynactin are

Tirumala et al. | Dynein regulation in vivo bioRχiv | 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.05.438428; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig. 3. Most dextran vesicles are stationary at a given time. a, SD microscopy image from a time-lapse of dextran vesicles in HeLa cells (left) and the corresponding kymograph (right). b, Plot of displacement vs. time of dextran vesicles in HeLa cells. c, Histogram with the duration of minus end-directed run on the x axis and probability on the y axis. d, SD + SRRF image of MTs (green) and dextran vesicles (magenta) in live cells. The white arrowheads indicate representative vesicles on the MT. e, SD + SRRF image of p62 (top left), dextran vesicles (top right) in live cells and their merge (bottom, p62 in green and dextran in magenta). The white arrowheads in the merged image indicate representative vesicles that colocalise with p62, and are shown as green and magenta arrowheads in the p62 and dextran images respectively. f, Histogram of the probability of cooccurrence of p62 with Rab5, indicating a high likelihood of dynactin being present in a complex with endosomal cargo. In a, d and e, ‘N’ marks the location/direction of the nucleus. in proximity to each other on the MT (Fig. 3e, f). tionary dextran vesicles started moving together with mDHC- Dynein molecules are activated upon attach- GFP towards the MT minus ends upon dynein binding (Fig. ment to dynactin and cargo on the MT 4a-d, n=8/9 events from N=2 independent experiments with Thus far, we discovered that while dynein transiently ~100 cells). interacted with the MT, dynactin and cargo appeared together and close to MTs. We therefore sought to understand how dynein interacted with the dynactin-cargo complexes Conclusions on MTs. First, we performed dual-colour imaging of dynein Taken together, we observed that: (i) single molecules of and dextran and observed that vesicles that had dynein signal dynein stochastically interacted with MTs. (ii) In some in- were more likely to move towards the minus end of MTs as stances, this attachment to MTs resulted in a successful en- expected (Extended Data Fig. 5e-g). Next, by comparing the counter with the dynactin-cargo complex and therefore, a mi- intensity of dynein on dextran vesicles to single molecule nus end-directed run that lasted about half a second. (iii) binding events, we estimated that, on an average, there were Dynein detached from the cargo to conclude the run (Fig. 1-2 dynein molecules bound to a dextran vesicle (Extended 4e). For long-range movement of endosomal cargo, these Data Fig. 5h). steps are required to be repeated multiple times. Such strate- Finally, to verify if single molecules of dynein could be acti- gies have been described in the past for transcription fac- vated and perform minus end-directed movement when they tors seeking speciﬁc sequences along the DNA (27, 28). stochastically bound to MTs and encountered dynactin-cargo Since cytoplasmic dynein is the only minus end directed complexes, we performed fast dual-colour HILO imaging of motor in many cell types, how dynein interacts with and single dynein molecules and dextran vesicles. To rule out transports different types of cargo is an interesting question. artefacts due to the presence of both untagged hDHC and Recent research suggests that cargo speciﬁc adaptors like mDHC in these cells, we performed these experiments in BicD2 (for Rab6-positive cargo), Hook1/3 (for Rab5-positive cells where we depleted hDHC. In these videos (Supplemen- cargo) have differential interaction with dynein (3). These tary Video 7), we observed instances where previously sta- cargo adaptors could modulate the interaction time of dynein

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RNAi experiments. For all RNAi experiments, cells were ﬁrst grown in glass-bottom imaging dishes (Cat. no. 81518, Ibidi) for 48 h, transfected with the appropriate concentration of siRNA using Jet Prime transfection reagent (Cat. no. 114, Polyplus) and imaged after 48 h. For hDHC RNAi the following siRNA sequence was used: 5’-ACA-UCA-ACA- UAG-ACA-UUC-A-3’10. For p150 RNAi the following siRNA sequences were used: 5’-GUA-CUU-CAC-UUG- UGA-UGA-A-3’, 5’-GAU-CGA-GAG-ACA-GUU-AUU- A-3’ (17). siRNA oligonucleotides were procured from Eurogentec, Belgium. To quantify the knockdown of proteins, the cells were lysed 48 hours after siRNA transfection to obtain protein solution and SDS-PAGE, western blotting was performed. The following primary antibodies were used to visualise proteins of interest and controls: Rabbit DYNC1H1 Polyclonal Antibody (PA5-49451, Invitrogen, 0.5 µg/mL), Rabbit Dynactin 1 Polyclonal Antibody (PA5- 37360, Invitrogen, 0.5 µg/mL), Mouse GAPDH Loading Control Antibody (MA5-15738, Invitrogen, 0.5 µg/mL), Rabbit Actin Polyclonal Antibody (ab8227, AbCam, 0.5 µg/mL). The following secondary antibodies were used: Anti-Rabbit HRP (31460, Invitrogen, 0.16 µg/mL) and Anti-Mouse HRP (62-6520, Invitrogen, 0.3 µg/mL).

Immunofluorescence. To immunostain p150 and tubulin, Fig. 4. Cargo capture and dynein activation are coupled a, Image of dynein cells were first grown for 48 h in glass-bottom imaging dishes (left), from a HILO microscopy time-lapse video and the corresponding kymograph (right). A representative binding event of a single dynein (green arrowheads) is (Cat. no. 81518, Ibidi) and fixed in ice-cold methanol at shown in the kymograph. b, Image of dextran (left) from the same HILO microscopy -20°C for 3 min. Cells were washed thrice in phosphate time-lapse video from which a was obtained and the corresponding kymograph buffered saline (PBS) for 5 min each and incubated in block- (right). A representative minus end directed run of the dextran vesicle (magenta arrowheads) is shown in the kymograph. c, Merge of images (left) shown in a, b ing buffer (5% BSA in PBS) for 60 min. Subsequently, the and the corresponding kymograph (right). The white arrow heads point to dynein cells were incubated with p150 and α-tubulin antibodies in and dextran moving together towards the minus end. d, Plot of displacement vs time antibody dilution buffer (1% BSA in PBS) for 60 min. Cells of the dextran vesicle (magenta) indicated in a, alongside the intensity of dynein on that vesicle (green), showing a short minus-end directed run of the vesicle upon were then washed thrice in PBS for 5 mins and incubated dynein-binding. e, Schematic of dynein’s cargo search strategy. In a, b and c, ‘N’ with secondary antibodies in antibody dilution buffer for 45 marks the location/direction of the nucleus. min. At the end of incubation with secondary antibodies, the cells were washed with PBS and imaged immediately. The molecules with the cargo thereby leading to different trajec- following primary antibodies were used. Rabbit Dynactin tories. Such stochastic binding and unbinding allow the same 1 Polyclonal Antibody (PA5-21289, Invitrogen, 2 µg/mL), dynein molecule to sample and interact with a wide range Mouse α Tubulin Monoclonal Antibody (32-2500, Invitro- of cargo, akin to round robin scheduling. Movement to the gen, 2 µg/mL). The secondary antibodies used were Don- plus or minus ends of MTs could therefore also be tuned by key Anti-Rabbit A555 Antibody (A31572, Invitrogen, 0.4 slightly increasing the bias in one direction, for example by µg/mL) and Goat Anti-Mouse A647 Antibody (A28181, In- increasing the number of adaptors for dynein on a cargo des- vitrogen. 2 µg/mL) tined towards the minus end. To immunostain p150 and p62, p62 and α-Tubulin and EB1 and α-Tubulin the following method was used. Cells were Methods first grown for 48 h in glass bottomed imaging dishes and Cell culture. HeLa cells were cultured in Dulbecco’s fixed in ice-cold methanol at -20°C for 3 min. Subsequently, Modified Eagle Media (DMEM) containing 100 units/mL the cells were washed thrice in PBS for 5 min each and in- Penicillin, 0.1 mg/mL Streptomycin, 2 mM L-Glutamine, cubated in antibody dilution buffer (2% BSA, 0.1% Triton- 400 µg/mL Geneticin (HiMedia Laboratories) and sup- X100, in PBS) for 10 min. Next, the cells were incubated in plemented with 10% fetal bovine serum (Sigma Aldrich). antibody dilution buffer containing the appropriate antibod- Cells were grown in an incubator at 37°C under 5% CO2. ies for 60 mins. Cells were washed thrice in PBS for 5 min During live-cell imaging, cells were maintained in live- each and incubated with appropriate secondary antibodies in cell imaging solution (140 mM NaCl, 2.5 mM KCl, 1.8 antibody dilution buffer for 30 min. At the end of incubation mM CaCl2, 1 mM MgCl2, 4 mg/mL D-Glucose, 20 mM with secondary antibodies, the cells were washed well in PBS HEPES) at 37°C using a stage-top incubator (Oko Labs). and imaged immediately. The following primary antibodies were used. Rabbit Dynactin 1 Polyclonal Antibody (PA5-

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21289, Invitrogen, 2.5 µg/mL), Rabbit Alpha Tubulin Mon- expressing low levels of mDHC-GFP. The diameter of the il- oclonal Antibody (PA5-22060, Invitrogen, 2 µg/mL), Mouse luminated area was kept constant at 30 µm and the long axis Dynactin 4 Monoclonal Antibody (MA5-17065, Invitrogen, of cell was aligned perpendicular to the excitation laser. Fi- 2 µg/mL) and Mouse EB1 Monoclonal Antibody (412100, nally, we adjusted the angle of incidence of the excitation Invitrogen, 2 µg/mL). The secondary antibodies used were laser, such that the fluorescent spots in a plane ~0.5 µm from Donkey Anti-Rabbit A647 Antibody (A32795, Invitrogen, 2 the cover slip appeared bright and distinct. The incidence an- µg/mL) and Goat Anti-Mouse A555 Antibody (A21422, In- gle and orientation of excitation laser beam were adjusted us- vitrogen. 2 µg/mL). ing the H-TIRF module and the laser power (50 mW at fiber) To immunostain Rab5 and p62, cells were first fixed in 4% was kept at 40%. To visualise single molecules of dynein, Paraformaldehyde (PFA) for 15 mins. The cells were washed time-lapse images were acquired at 50 fps with 20 ms expo- well in PBS and incubated for 45 min in antibody staining sure per frame. solution (0.2% Saponin, 0.1% BSA and 0.02% Sodium To improve the signal-to-noise ratio, a five-frame sliding av- Azide in PBS) containing primary antibodies against Rab5 erage of the time-lapse images was used for particle track- and p62. Subsequently, cells were washed well in PBS and ing. In these videos, the appearance and subsequent disap- incubated for 45 min in antibody staining solution containing pearance of intensity at a particular location was classified as appropriate secondary antibodies. Cells were washed well in a binding event. Such binding events were visually identi- PBS and imaged immediately. The following primary anti- fied and tracked from the start to end frame using Low Light bodies were used. Rabbit Rab5 Monoclonal Antibody (3547, Tracking tool (30) in Fiji/ImageJ (31). The fluorescent spots Cell Signaling Technology, 14 µg/mL), Mouse Dynactin 4 were classified as stationary, minus-end moving or plus-end Monoclonal Antibody (MA5-17065, Invitrogen, 2 µg/mL). moving by marking a region close to the nucleus as the mi- The secondary antibodies used were Donkey Anti-Rabbit nus end and calculating the displacement of fluorescent spots A647 Antibody (A32795, Invitrogen, 2 µg/mL) and Goat with respect to the minus end. Particles moving away from Anti-Mouse A555 Antibody (A21422, Invitrogen. 2 µg/mL). the nucleus for >0.32 µm were classified as plus-end moving, and particles with displacement >0.32 µm towards the Dextran uptake. To visualise fluorescent dextran vesicles nucleus were minus-end directed, and particles with net dis- HeLa cells that were grown in glass-bottom imaging dishes placement <0.32 µm were classified as stationary. (Cat. no. 81518, Ibidi) for 48 h were transferred to serum- For dual channel imaging of dynein and dextran, free DMEM for 4 h in a 37°C CO2 incubator. Subsequently, videos were acquired at 20 fps with 25 ms expo- the cells were pulsed for 10 min in complete DMEM sure per channel. To observe dynein-dextran interac- containing 200 µg/mL A647-Dextran (D-22914, Invitrogen). tion with a better signal-to-noise ratio, a two-frame Cells were then washed well with live-cell imaging solution sliding average of time-lapse images was used. before proceeding for microscopy. In all experiments, imaging was completed within 45 min of dextran uptake. Spinning disk (SD) confocal microscopy and image analysis. To quantify the correlation between expression level of Transfection. To visualise fluorescently tagged proteins, dynein and its clustering, z-stack images of live HeLa cells cells were transfected with the appropriate plasmids using expressing mDHC-GFP were acquired using a 60x, 1.4 NA Jet Prime Transfection Reagent (Cat. no. 114, Polyplus). objective with 50 ms exposure. The intensity comparison 3 h after transfection, the cells were washed well in PBS, was done for the lowest plane at which clusters were dis- grown in complete DMEM at 37°C and imaged ~20 h tinctly visible. The dynamics of mCherry-p150 in cells were later. The following plasmids were used in this study. (i) visualised by acquiring 60-s long movies of the cell with a mCherry-tubulin was a gift from Mariola Chacon, TUD, 1.4 NA 60x objective, 100 ms exposure per frame and 1 s Dresden. (ii) Gal4T-mCherry was a gift from Thomas interval between frames. The dynamics of dextran vesicles Pucadyil, IISER Pune. (iii) mCherry-DCTN1 was a gift from along the MTs was visualised by acquiring 30-s long movies Prof. Kozo Tanaka, Tohoku university, Japan. (iv) mCherry- of the cell with a 60x, 1.4 NA objective, 100 ms sequential Rab5 was a gift from Gia Voeltz (Addgene plasmid - 49201). exposure/channel and 1 s interval between frames. The dynamics of dextran vesicles was visualised by acquiring a 10-s Microscopy. All imaging was performed on a Nikon Ti2E long movie with 100x 1.49NA TIRF objective, 100 ms ex- inverted microscope equipped with a Toptica MLE laser com- posure/frame and no interval between frames. The move- biner, Nikon H-TIRF Module, Yokogawa CSU-X1 spinning ment of dextran vesicles was classified in the following way: disk module, an Andor iXon 897 EMCCD camera and an the movement between two consecutive frames was classi- Oko Lab stage top incubator. The microscope was controlled fied as zero if it was less than 50 nm (the tracking accuracy of using Nikon NIS Elements software or Micromanager (29). low light tracking tool under the imaging conditions used30). Then, displacement for four consecutive frames towards or HILO microscopy and particle tracking. For HILO mi- away from the nucleus was classified as a minus or plus end- croscopy, a Nikon 100X 1.49NA TIRF objective was used. directed run. We optimised the HILO microscopy setting for each cell: The SD+SRRF images were obtained by taking the mean of First, to avoid overexpression artefacts, we visualised cells the radiality map of 100 images of a single field acquired

6 | bioRχiv Tirumala et al. | Dynein regulation in vivo bioRxiv preprint doi: https://doi.org/10.1101/2021.04.05.438428; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

under a 100x, 1.49NA objective with the SD confocal mi- 3. R. J. McKenney, W. Huynh, M. E. Tanenbaum, G. Bhabha, and R. D. Vale. Activation croscopy setup. The radiality magnification in SRRF was set of cytoplasmic dynein motility by dynactin-cargo adapter complexes. Science, 345(6194): 337–341, jul 2014. ISSN 0036-8075. doi: 10.1126/science.1254198. to 4 for all experiments. To visualise the association between 4. Max A Schlager, Ha Thi Hoang, Linas Urnavicius, Simon L Bullock, and Andrew P Carter. In MTs and dextran vesicles in live cells, the SRRF acquisition vitro reconstitution of a highly processive recombinant human dynein complex. The EMBO Journal, 33(17):1855–1868, sep 2014. ISSN 0261-4189. doi: 10.15252/embj.201488792. settings used were 20ms exposure, ring radius of 0.5 for MTs 5. Kai Zhang, Helen E. Foster, Arnaud Rondelet, Samuel E. Lacey, Nadia Bahi-Buisson, and 3 for dextran. To visualise p62 with tubulin and EB1 Alexander W. Bird, and Andrew P. Carter. 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